Correlation between circulating ANGP T2 levels and Ki67 LI (i.e., % of Ki67‐positive cells). All box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median. Results are expressed as mean ± SEM. **P = 0.014 by one‐way ANOVA (P = 0.007 by Kruskal–Wallis test).

Expression of ANGPT1 and ANGPT2 in human unaffected anterior pituitary (control, n = 3) and in human NF‐PitNETs. Thirty‐seven human NF‐PitNETs were analyzed for ANGPT2 expression and 14 for ANGPT1. NF‐PitNET panels show representative cases. Original magnification: 400×; scale bar: 20 µm.

Summary of the IHC results for human NF‐PitNETs.

Expression of Tie2 receptor in human normal pituitary (n = 3) and human NF‐PitNET tissues (n = 10). Original magnification: 400×; scale bar: 20 µm.

Quantification of tumor‐induced vessel sprouting in zebrafish embryos engrafted with GH3 ‐shCtrl versus shC2 cells (n = 20 each). Data were normalized against the mean of the control (shCtrl) group arbitrarily set to 1. A.U. arbitrary Units; *P = 0.0319 (t‐test).

Example of flow cytometry data of primary rat pituitary cells from a tumor‐bearing MENX rat gated for cell surface Tie2 and CD31 expression (cells were pre‐gated for Tie2).

Percentage of Tie2⁺CD31⁻ (PitNET cells) and Tie2⁺CD31⁺ (ECs) in the pituitary glands of 7 age‐matched (8 months) MENX rats. Data are expressed as the mean ± SEM. ****P < 0.0001 (t‐test).

A representative graph displaying the Tie2 fluorescence intensity and counts of Tie2⁺CD31⁻ and Tie2⁺CD31⁻ cell populations the pituitary of one rat.

Fluorescence intensities of the Tie2⁺CD31⁻ and Tie2⁺CD31⁻ cell populations across all 7 pituitary samples. Data are expressed as the mean ± SEM. n.s, not significant (Mann–Whitney test).

Quantification of the interactions between Angpt2 and Tie2 in rat PitNET tissues versus negative controls obtained using only 1 antibody (Appendix Fig S7C). Box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median, whereas the “x” represents the mean.

PLA was performed as in (A) on isolated rat primary NF‐PitNET stimulated with rhANGPT2.

Quantification of Angpt2/Tie2 interactions in primary NF‐PitNET incubated with/without rhANGPT2. The total number of cells counted is reported in parenthesis (i.e., 212 and 345) as well as the number of positive cells (= showing at least one interaction). The graphs show the percentage of cells having the number of interactions reported on the x axis. Box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median, the “x” represents the mean, and the circle outlier points.

Expression of total Fak and P‐Fak (Y397) in serum‐starved GH3 Ctrl‐KO and Tie2‐KO clones #18 and #19 stimulated with rhANGPT2 for 30 min or left untreated. The numbers represent the ratio phospho/total Fak. Anti‐α‐tubulin antibody was used to check for equal loading. Blot shown is representative of 3 independent experiments.

Scheme of the in vivo study in mouse xenografts of GH3 Ctrl‐KO and Tie2‐KO cells (clone #19).

T2‐weighted images of two xenografted tumors taken at day 0 and 21 (largest diameter) representing the two animal groups. Scale bar: 2 mm, except GH3‐Ctrl KO day 21: 4 mm.

Changes in tumor volumes as determined by MRI were normalized to the day 0 value (=100%) for each animal. All box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median. **P = 0.003 (one way ANOVA).

Expression of Tie2 (red) in the xenografted tumors (n = 3 each group). Nuclei were counterstained with DAPI (blue). Original magnification: 400×; scale bar: 50 µm.

T2‐weighted images of two xenografted tumors taken at day 0 and 21 (largest diameter). The tumors are representative of the two treatment groups Scale bar: 2 mm.

Changes in tumor volumes as determined by MRI were normalized to the day 0 value (= 100%) for each animal. All box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median. **P = 0.007 (one way ANOVA).

Changes in tumor volumes in female rats treated with AMG386 (n = 6) or with placebo (n = 5) for 14 days as determined by anatomical MRI, normalized to the day 0 value. Box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median. P = 0.0673, not significant (one way ANOVA).

ADC values of the rat PitNETs before (day 0) and after (day 14) treatment with AMG386 (red, n = 8) or placebo (blue, n = 5). Box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). The line in the box represents the median. **P = 0.009; n.s., not significant (t‐test).

Ex vivo expression of Tie2, P‐Tie2, Ki67 and Annexin V in PitNETs of female rats treated with AMG386 or placebo for 14 days. Original magnification: 200×; scale bar: 50 µm. Panels shown are representative of the two animal groups.

Quantification of Annexin V intensity from tissues stained as in (F) (n = 4/group). Three different areas per tumor sample were analyzed. Intensities are expressed as arbitrary units ± SEM. ***P < 0.001 (by t‐test).

IHC was performed on pituitary tissues from wild‐type (n = 5) and MENX mutant rats (n = 10) using antibodies against Angpt1, Angpt2. Representative stainings are shown. Original magnification: 400×; scale bar: 20 µm. N, normal area; T, tumor area.

Expression of Angpt2, Tie2, and CD‐31 in rat NF‐PitNETs and associated ECs (used as positive control). Consecutive tissue sections of rat NF‐PitNETs (n = 4) were stained with the indicated antibodies. One representative tumor is shown. The dashed shape indicates the vessel present in the consecutive slides. Original magnification: 200×; scale bar: 20 µm.

Immunofluorescence (IF) of GH3 cells for Angpt2 (red) and Tie2 (green). Nuclei were counterstained with DAPI (blue). Original magnification: 400x; scale bar: 20 µm. Panels shown are representative of 3 independent experiments.

Cell proliferation of GH3 cells transfected with siAngpt2 or scrRNA POOLs and incubated with rhANGPT2 (+rhANGPT2) or left untreated (−rhANGPT2) normalized against scrRNA‐transfected cells. ***P < 0.0001 (one‐way ANOVA).

In samples parallel to (C), activated caspase‐3/7 was measured to assess for apoptosis. ***P < 0.0001; *P = 0.0411 (one‐way ANOVA).

Cell proliferation of GH3 cells transfected with the individual siAngpt2 or scrRNA and incubated with rhANGPT2 (+rhANGPT2) or left untreated (−rhANGPT2) normalized against scrRNA‐transfected, untreated (scr‐rhANGPT2) cells. n.s., not significant; ***P < 0.0001; **P < 0.002 (one‐way ANOVA).

In samples parallel to (E), activated caspase‐3/7 was measured to assess for apoptosis. n.s., not significant; ***P < 0.0001 (one‐way ANOVA).

Cell proliferation of GH3‐shAngpt2 (#2) cells or cells transduced with shCtrl was measured 24h later and normalized against shCtrl. ***P < 0.0001 (t‐test).

In samples parallel to (G), activated caspase‐3/7 was measured to assess for apoptosis. ***P < 0.0001 (t‐test).

GH3 cells transfected with siAngpt2 or scrRNA POOLs were incubated with conditioned medium (CM) from rat primary PitNET cells for 24 h. CM was pre‐incubated with AMG386 (red bars), or left untreated (blue bars). Cell proliferation was measured and normalized against that of scrRNA‐transfected cells. n.s., not significant; ***P < 0.0001; **P = 0.00116; *P = 0.0192 by two‐way ANOVA.

Cell viability of isolated rat primary EC cells incubated with CM from isolated rat primary PitNET cells. The experiments was performed independently 2 times, each with 3 technical replicates. Results are expressed as mean ± SEM. *P < 0.0368 (t‐test). (C–I) Data are expressed as the mean ± SEM of three biological replicates, each with 3–6 technical replicates.

Quantification of P‐Tie2 immunostaining intensity in cells shown in (A) shCtrl‐transduced GH3 cells (266.71 ± 12.12); in shAngpt2 cells (176.04 ± 8.46); in control cells treated with rhANGPT2: shCtrl + rhANGPT2: 246.33 ± 9.79 (versus shCtrl, not significant by t‐test); in treated knockdown cells: shAngpt2 + rhANGPT2: 218.68 ± 18.16 (versus shC2; *P = 0.0421 by t‐test). The experiment was performed twice each with three technical replicates. Intensities are expressed as arbitrary units ± SEM. ***P < 0.0001 by t‐test.

Tie2 and P‐Tie2 expression in serum‐starved GH3 cells transfected with siAngpt2 POOLs and stimulated with rhANGPT2 for the indicated times.

Tie2 and P‐Tie2 expression in cells as in (C) stimulated with the indicated doses of rhANGPT2 for 30 min.

Co‐IF for Angpt2 (red) and P‐Tie2 (green) of a representative rat primary NF‐PitNET. Nuclei were counterstained with DAPI. White arrows point to Angpt2‐positive (P‐Tie2 negative) cells in adjacent non‐tumor area. Scale bars: 50 µm. T, tumor area.

Cell viability of rat primary PitNET cells (R‐PitNET) treated with AMG386 (n = 5 rats) (C) or Tie2‐KI (n = 4 rats) (D) or left untreated (ctrl) normalized against untreated cells. Primary cultures were treated, each with 6 technical replicates. Results are expressed as mean ± SD. ***P < 0.0001 (t‐test).

Expression of phosphorylated (P) and total Akt, p38 and Erk1/2 in two R‐PitNET cultures with enough cells upon treatment with AMG386.

Expression of phosphorylated (P) and total Akt in one R‐PitNET culture treated with Tie2‐KI or left untreated (ctrl).

Cell viability of human primary PitNET cultures treated with (G, H) AMG386 (n = 16) or (I, J) Tie2‐KI (n = 12) normalized against untreated control cells for each patient (set to 1). Significance in (G, I): *P < 0.05; **P < 0.01; ***P < 0.001 (by t‐test, comparing each treated sample with its untreated control). Results in (H) and (J) are expressed as mean ± SEM of the normalized values of 6 technical replicates for each samples. Each dot represents the relative cell viability of one technical replicate. Significance in (H, J) ***P < 0.0001 (t‐test).

Example of responsive H‐PitNET treated with AMG386 or Tie2‐KI. Total proteins were extracted and probed for P‐ and total Akt and Erk1/2. Anti‐α‐tubulin antibody as used to check for equal loading. The blot shown is representative of three independent cultures.